Antibiotic 1745A/X and methods for the production thereof

ABSTRACT

A new antibiotic, designated as antibiotic 1745A/X, is produced by the fermentation of the known microorganism Streptomyces antibioticus, ATCC 11891, using the novel compound erythronolide A oxime as the substrate. This new antibiotic is useful as an antibacterial agent.

United States Patent [191 Kierstead et al.

[ ANTIBIOTIC 1745A/X AND METHODS FOR THE PRODUCTION THEREOF [75] Inventors: Richard Wightman Kierstead;

Ronald Andrew LeMahieu, both of North Caldwell; David Pruess, Passaic, all of NJ.

[73] Assignee: Hoffmann-La Roche Inc., Nutley,

[22] Filed: Feb. 4, 1974 [2]] Appl. No.: 438,987

[52] US. Cl 260/343; 260/210 E; 424/180 [51] Int. Cl. C07D 313/00 [58] Field of Search 260/210 E, 343

[56] References Cited UNITED STATES PATENTS 3,361,737 1/1968 Jones 260/210 E Dec. 23, 1975 3,478,014 11/1969 Djokic et al 260/210 E 3,674,773 5/1972 Kurath 260/210 E 3,697,547 10/1972 Kurath et al 260/343 OTHER PUBLICATIONS Wagner and Zook, Synthetic Organic Chemistry, p. 739, Wiley and Sons, Inc., New York, 1953.

Primary Examiner-Johnnie R. Brown Assistant Examiner-Cary Owens Attorney, Agent, or Firm-Samuel L. Welt; Bernard S. Leon; Frank P. Hoffman 2 Claims, 2 Drawing Figures US. Patent Dec. 23, 1975 Sheet10f2 3,928,387

AONVLLIWSNVELL LNBOHBd WAVE NUMBERS IN cm-' US. Patent Dec. 23, 1975 Sheet 2 of2 3,928,387

first converting the oxime to the corresponding N- ANTIBIOTIC l745A/X AND METHODS FOR THE Oxide f the f l PRODUCTION THEREOF DETAILED DESCRIPTION OF THE INVENTION 5 This invention relates to a new antibiotic and to methods for its production by fermentation, its isolation and purification, and its use an antibacterial agentv The present invention includes within its scope the antibiotic l745A/X in pure form, in dilute forms, and as a crude concentrate.

The new antibiotic, designated hereinafter as antibiotic 1745A/X, is produced by the fermentationof the known microorganism Streplomyces antibioticus, ATCC 11891, using as the substrate the novel compound erythronolide A oxime. The species of Streptomyces described herein and identified as Streptomyces antihiuticus, ATCC 1 1891 includes all strains of Streptomyces which when fermented under the conditions described herein produce antibiotic l745A/X and which cannot be definitely differentiated from the strain ATCC l l89l and its subcultures, including mutants and variants. By the term mutants as used herein, there is intended mutants produced from the described organism by various means such as chemical mutagenic agents, ultra-violet radiation, X-radiation,

Conversion of the oxime of formula I above to the corresponding N-oxide of formula II is accomplished phage exposure and the like. by treating the oxime with hydrogen peroxide. This As described above, antibiotic l745A/X is produced r tion is pr f rably effe m the pr nce of n by the fermentation of Streptrmzyces antibi0tl'cus,ATCC melt orgarc ol n uit l ol ents Inclu e lower 11891, si g as the sub t t i th f r t ti th 3( alkanols such as methanol, ethanol, propanol and the novel compound erythronolide A oxime, This comlike. It is expedient to effect this reaction at a tempera pound is prepared by the cleavage of the cladinose and ture in the range of from 0 to about C, with room desosamine sugar moieties from the known oxime of of temperature being preferred.

erythromycin A of the formula The so-obtained N-oxide of formula II is then con- N (CI-I3) 2 I O CH 3 /CII 3 ocu Cleavage of the cladinose and desosamine sugar moiverted to the corresponding des-( dimethyamino-N eties from the oxime of formula I is accomplished by oxide) compound of the formula Cleavage of the cladinose and the modified desosamine sugar moieties from the compound of formula III is accomplished by treating said formula III compound with a mineral acid. Suitable mineral acids for this purpose include hydrochloric acid, hydrobromic acid and sulfuric acid. This reaction is preferably effected in the presence of an inert organic solvent. Examples of solvents that can be employed for this reaction include lower alkanols such as methanol, ethanol, propanol and the like. Temperatures in the range of from about to about 30 C can be employed for the reaction, with room temperature being preferred.

The compounds of formulae II, III and IV are novel and as such form a part of the present invention. I

Cultivation of the organism Strepmnri'ces antibiotic'us, ATCC l 1891, using erythronolide A oxime as the substrate to produce the desired antibiotic l745A/X may be carried out utilizing a variety of fermentation techniques. In general, the following basic techniques can be employed in both flask and tank procedures. In

4 tinomyces Beer Medium VI broth (J. Appl. Micro bi01., 9, 394-399, 196] distributed by Difco Laboratories, Detroit, Mich. After incubation in the inoculum medium for up to three days small samples of the broth are transferred to .theculture medium where they are incubated at abou' t 28C on a rotary shaker. After incubation for up to" IOho'urs, erythronolide A oxime is added to the culture medium 'as'the'substrate and incubation is continued for from about 1' to 5 days. Whole broth samples are aseptically removed periodically for determination of the course of fermentation. For preparation of larger volumes of broth, inoculum is first prepared in 6 liter Erlenmeyer shake flasks or in 5 gallon pyrex bottles, fitted for aeration, sampling, etc. This broth is then transferred to the tank fermentors. Aeration in bottles and tanks is provided by forcing sterile air through the fermenting medium. In tanks, further agitation is provided by mechanical impellers. Antifoam agents such as lard oil, soy bean oil, silicone surfactants such as SAG No. 4130 (Union Carbide Corp.) and DCAF (Dow-Corning) are added as needed to control foam.

Slrepl0H1 \C.s'(IIIIibiOIiCUS, ATCC 11891 may be cultured in a variety of liquid culture media. Media which are especially useful for the purposes of the present invention include an assimilable carbon source such as starch, glucose, sucrose, molasses, glycerol, dextrin, corn starch, and the like, and an assimilable nitrogen source such as protein, protein hydrolysate, polypep tides, amino acids, corn steep liquor and the like. Complex nitrogenous material from varying sources will support production of antibiotic l745A/X. Examples of such-include plant materials such as soybean flour, animal materials such as meat meal digest, and microbial materials such as yeast. Inorganic cations and anions, such as potassium, sodium, calcium, magnesium, sulfate, phosphate, chloride, etc. can also be added to the culture medium. In addition, those elements such as cobalt, copper, iron, molybdenum, boron, etc. can be added if they are not supplied as impurities of other constituents of the med'n.

the flask fermentation, a loopful of spores from an agar slant of the culture is inoculated into 100 ml. of nutrient medium in a 500 ml. Erlenmeyer flask and incubated at about 28C on a rotary shaker for up to 3 days. The inoculation nutrient medium contains a nitrogen source, preferably selected from an acid or enzyme hydrolyzed protein source such as enzyme hydrolyzed milk products, enzyme hydrolyzed bean meal products and the like; a carbohydrate source such as glucose, sucrose, molasses, and the like; and inorganic salts such as phosphate and sulfate salts, sodium chloride, and the like. A preferred inoculum medium is the thermoac- The activity of antibiotic l745A/X can be measured in vitro by its zone of inhibition against the microorganism Sarcina lutea PCI in the standard paper-disc agar diffusion method. Inoculum was prepared from Sarcina lutea PCI grown for 20-24 hours in ml of broth containing Bacto-peptone 6g/l, cerelose l'g/l, N-Z amine A 4g/l, yeast autolysate 3g/] and beef extract 1.5 g/l. in a 500 ml cotton-plugged Erlenmeyer flask at 35C on a rotary shaker at 240 RPM. A portion of the cells were inoculated into molten agar (containing the same ingredients as the above-defined broth with the addition of agar l5 g/l). The molten agar was then poured into either 16 X 35.5 cm steel covered glass assay pans or 12 X 88 cm plastic petri dishes and allowed to solidify. Broth samples or partially purified samples of antibiotic l745A/X were applied to 6.5 mm diameter discs which were then placed on the agar.

' After incubation overnight at 37C the diameters of the zones of inhibition were measured. The zone diameters were found to be proportional to the log of the concentration between 3.0 and 100 mcg. per ml. of antibiotic l745A/X. A two-fold increase in the concentration of antibiotic l745A/X increased the zone diameter by 2.7 mm.

After fermentation is complete, a variety of procedures can be employed for the isolation and purification of antibiotic l745A/X. Suitable isolation and purification procedures include solvent extraction techniques, such as batchwise extraction or countercurrent continuous flow liquid-liquid extraction columns, and gel permeation chromatrography. In a preferred process, antibiotic 1745A/X is recovered from the culture medium by separation of the mycelium and any undissolved solids from the fermentation broth by conventional means such as by filtration or centrifugation. Antibiotic 1745A/X is then extracted from the filtered or centrifuged broth using either batchwise or counter current distribution extraction techniques. The solvent extraction may be performed using a pH range of from 8 to and employing as the solvent an inert organic solvent. Suitable solvents include alcohols, such as methanol, ethanol and the like, chlorinated hydrocarbons such as chloroform, methylene chloride and the like, ethyl acetate, butyl acetate, amyl acetate, acetone and acetonitrile, with methylene chloride being preferred. Final purification of antibiotic l745A/X can be achieved by chromatography on permeable gels. This purification technique is accomplished by percolation of prepurified preparations of the antibiotic, for example, preparations obtained by solvent extraction tech niques, through cross-linked polymerized dextran gels. In a preferred aspect of this purification technique, the pre-purified antibiotic preparation is chromatographed on Sephadex G- eluting with n-butanol/acetic acid/- water.

After filtration or centrifugation of the fermentation medium, thin layer or paper chromatography techniques can be employed to analyze for antibiotic 1745A/X. In addition, bioautography can also be employed advantageously.

The novel antibiotic of the invention, antibiotic 1745A/X, upon purification exists in crystalline form, m.p. l46l49C. From elemental analysis of the free base and the methiodide salt of antibiotic 1745A/X, the formula C H N 0 was assigned to the antibiotic. The antibiotic contains the elements carbon, hydrogen, nitrogen and oxygen in substantially the following percentages by weight:

Antibiotic l745A/X The following are various physical characteristics of antibiotic 1745A/X:

The infrared absorption spectrum of antibiotic 1745A/X 4% chloroform solution is shown in FIG. 1. The antibiotic exhibits characteristic absorption in the infrared region of the spectrum at the following wave lengths expressed in reciprocal centimeters:

Broad band at 3500 Complex absorption at 3000 Prominent bands at 1735, 1470, 1180, 1120, 1060 and 1000.

The low resolution mass spectrum of antibiotic 1745A/X shows a molecular ion at m/e 734 and major fragment peaks at 701, 649, 617, 573, 560, 546, 462, 416, 398, 251,174, 158 and 145.

The nuclear magnetic resonance spectrum of antibiotic 1745A/X is shown in FIG. 2. The NMR spectrum was obtained using CDCL, as the solvent and tetrame-- thylsilane (TMS) as the internal standard. The NMR spectrum exhibits prominent signals at 3.428, 2.288, 1.168 and in the region between 0.8 and 1.78.

TABLE 1 In Vim) Animicrobial Spectrum Antibiotic l745A/X Diameter of Inhibition Zones in mm. (Antibiotic 1745A/X in 3A alcohol -discs dipped and air dried) Test Organism* l0 meg/ml mcg/ml lmg/ Bacillus .tu/uilix 0 I2 23 Surt'inu lulcu 8 I9 28 M \'t'ululz'u'rimn [Ill/0i 0 l2 l9 Bur'iI/ux SI). 1283 B(TA) (J 13.5 22

*Microorganism grown in broth containing llactopeptonwhg/L, ccrclosclg/L, N-7. aminc A-4g/l. yeast autul \'sntc-3g/l. and bccfcxtracl -1.5g/l.

As indicated above, antibiotic 1745A/X is useful as an antimicrobial agent. Thus, this compound can be used as a medicament. For example, it can be used in the form of pharmaceutical preparations which contain it in admixture with a pharmaceutical organic or inorganic carrier material which is suitable for enteral or parenteral application such as, for example, water, gelatin, lactose, starches, magnesium stearate, talc, vegetable oils, gum arabic, polyalkylene glycols, vaseline, etc. The pharmaceutical preparations can be prepared in solid form (e.g., as tablets, dragees, suppositories, capsules) or in liquid form (e.g., as solutions, suspensions or emulsions). They may be sterilized and/or contain additives such as preserving, stabilizing, wetting or emulsifying agents, salts for varying the osmotic pressure or buffers. They can also contain other thera peutically valuable substances. Antibiotic 1745A/X can be administered at dosages adjusted to individual requirements and fitted to the pharmaceutical exigencies of the situation.

The following examples are illustrative but not limitative of the present invention. All temperatures given are in degrees Centigrade.

EXAMPLE 1 Preparation of Erythromycin A oxime N-oxide To 53.6 g (0.07 mole) of erythromycin A oxime [Massey et al., Tetrahedron Letters, 157 (1970)] in 2150 ml methanol was added 1300 ml 1.1 mole) of 3% H 0 and the solution was left at room temp for 19 hr. Most of the methanol was removed in vacuo and the crystalline product which separated was filtered and air dried. Tlc showed a single spot slower moving than the starting material. Recrystallization from MeOH-H O yielded the above-named product, mp 181-183.

EXAMPLE 2 Preparation of 3'-De(dimethylamino)-3 ',4'-dehydroerythromycin A oxime Erythromycin A oxime N-oxide (5.00 g, 6.5 mmole) was pyrolyzed for 3 hr at l55/0.1 mm in a slowly spinning flask. Tlc revealed a major spot faster moving than the starting material along with two minor impurities. The product was dissolved in methanol, decolorized with charcoal and crystallized from MdOH-H O to yield the above-named product, mp l58.

EXAMPLE 3 Preparation of Erythronolide A oxime A solution of 22.55 g. (0.032 mole) of 3-de-(dimc thylamino)-3,4-dehydroerythromycin A oxime in 1.5 l of 3% HCl in methanol was left at room temp for 21 hr. The methanol was removed in vacuo, EtOAc was added to the residue and the solution was washed with dilute NaHCQ, solution. After drying, the solution was stirred briefly with charcoal and concentrated to dryness. Tlc revealed a major spot much slower moving than the starting material, several minor impurities and two fast moving spots. Two crystallizations from (CH;,) CO-C,;H gave the above-named product, mp 236239.

EXAMPLE 4 Preparation of Antibiotic l745A/X Srreptonrvces untibiulicus, ATCC 1 1891, was streaked on slants of SP5 agar (yeast extract lg., beef extract 1g., tryptose 2g, FeSO, trace, glucose lg., agar 15g, distilled water 1 liter-pH adjusted to 7.2) and incubated at 28 for days. One slant was then washed with ml of thermoactinomyces Beer Medium Vl (J. Appl. MicrobioL, 9, 394399, 1961) and 3 ml of this spore suspension was inoculated into 100 ml. of the same broth in cotton plugged 400 ml. Erlenmeyer flasks. These were grown at 28 and 250 RPM on a 2 inch stroke New Brunswick rotary shaker for 2 days. The flask contents were pooled, and 200 ml was inoculated into a stirred jar containing 10 l. fermentation broth (cornstarch l5.2g., soybean meal 15.2g., cornsteep liquor l0g., NaCl 3.3g, CaCo;,5g., distilled water 1,000 ml., pH adjusted to 6.8). The silicone antifoam agent SAG No. 4130 (Union Carbide) was added intermittently throughout the course of the fermentation to suppress foaming. After 6 or 7 hours of growth, lg. of Erythronolide A oxime dissolved in 100 ml 3A alcohol was added to the fermentation and incubation was continued as before.

The course of the fermentation was followed by measuring the antibiotic potency of supernate samples with an antibiotic standard curve on Sarcina [urea test plates and also by thin layer chromatography of methylene chloride extracts of supernate samples (after adjustment to pH 8.5) on silica gel plates run twice through a solvent system of CHgClz, 90/95% aqueous MeOH, lO/conc. NH OH, 1.These plates were bioassayed on Bacillus sp. 1283B in prepared agar (Bactopeptone- 6g/l., cerelose-lg/L, N-Z amine A-4 g/l., yeast autolysate-3g/l., beef extract-1,5g/l and agar g) by inverting the plate for one-half hour on the agar, removing the plate, and incubating the agar pan overnight at 37.

Antibiotic zones were distinguished by areas of no growth on the agar. The same plate could also be sprayed with a spray of 0.15% xanthydrol in a 12:1 mixture of concentrated hydrochloric acid and glacial acetic acid and heated for 5 minutes at 120. During fermentation of the microorganism Srreptomyces antz biotic-us ATCC l 1891, under the conditions described above, this microorganism co-produces the known antibiotic oleandomycin and antibiotic 1745A/X. If the silica gel plates are sprayed with xanthydrol, the antibi otics appear as dark pink spots against a light pink background. In this fermentation, the oleandomycin produced by the culture had an R; of about 0.7 and antibiotic l745A/X showed an R, of about 0.5. The potency of the supernate was usually about 808/ml between 40 and 64 hours after dosing, at which time the jar was harveste The contents of the jar was centrifuged to remove the cells and the supernate was adjusted to pH 8.5 with NaOH. The supernate was extracted twice with equal volumes of CHQCI which were then pooled and evaporated below to about 1 liter. This concentrate was extracted three times with l/2 volume of 0.01M H PO, and the CH Cl phase discarded. The aqueous phase was immediately adjusted to pH9 with KOH and NaCl was added almost to saturation. This was then extracted twice with one-half volumes of CH Cl The combined organic phases were evaporated under reduced pressure to yield 1.2g of solids.

Chromatography was effected by swelling 115g of Sephadex G-25 superfine beads in the lower phase of a solvent system consisting of n-butanol/acetic acid/water: 4/1/5. The Sephadex was poured into a column, 3.8

cm ID by 68 cm, and 3 bed volumes of upper phase of the above solvent system was passed through the column. A solution of 0.56g of the solids, obtained as described in the preceding paragraph, in lml. of the upper phase of the solvent mixture was applied to the column and development was effected in the same upper phase. An active fraction was obtained at an elution volume of 300-360 ml. The solvent was removed under reduced pressure to yield 152 mg of solids. These solids were dissolved in 10 ml. water and the pH was adjusted from 5.2 to 9.4 with 0.5 N NaOH.

The solution was extracted three times with 10 m1 of ethyl ether and the combined organic phases were evaporated under reduced pressure to yield 1 15 mg of solids. The solids were dissolved in 2 ml of ether and 3 ml of hexane was added. The solvent was partially evaporated under reduced pressure until the solution became turbid. After maintaining the solution at 20, 57 mg. of antibiotic 1745A/X as a semi-crystalline precipitate was obtained. This material was recrystallized from ethanol/water: lO/90. Bioautography of a thin layer chromatogram of these crystals indicated only one active component was present.

EXAMPLE 5 Purification of Antibiotic l745A/X Using Counter-current Distribution Following the procedures described in Example 4- above, several fermentations of total volume 290 l to tracted three times with one-third volumes CH Cl The combined organic solvent extracts were evaporated under reduced pressure to yield 14 g solids.

Counter-current distribution in a 100 tube apparatus m1 tube volume) was effected by charging 3g of the solid into the 1st three tubes. The solvent system con sisted of the upper and lower phases obtained from the mixture n-butanol/acetic acid/waterzlO/l/lO adjusted to pH 2.95 with 2N NaOH. The activity was obtained from tubes 78-88 and the solvent mixture partially evaporated under reduced pressure to 200 ml. The pH was adjusted to 9.5 with 40% NaOH and the solution extracted four times with one-half volumes of CHzClg. The combined organic phases were evaporated under reduced pressure to yield 0.60 g solids. Crystallization was effected from ethanol ether to yield antibiotic 1745A! m n 146 40 Example 6 Example 9 Tahlct Formulation Per Tablet Capsule Formulation Per Capsule Antibiotic l745A/X 500 mg Corn Starch 30 mg Antibiotic l745A/X g Lactose XX mg Lactose USP 125 mg Ge ati 13 g Corn Starch. USP 30 g Talcum mg T t USP 5 mg Magnesium Stcarate 5 mg T l W i ht 210 mg Total Weight 650 mg PROCEDURE:

Example 7 Tablet Formulation Per Tablet Antibiotic l745A/X 100 mg Lactose. USP 202 mg Corn Starch. USP so m Amijel BO 1 1* mg Calcium Stearate 8 mg Total Weight 410 mg PROCEDURE:

1. Antibiotic 1745A/X lactose, corn starch, and Ami jel BO 11 were blended in a suitable mixer.

2. The mixture was granulated to a heavy paste with water and the moist mass was passed through a No. 12 screen. It was then dried overnight at 110F.

3. The dried granules were passed through a No. 16 screen and transferred to a suitable mixer. The calcium stearate was added and mixed until uniform.

4. The mixture was compressed at a tablet weight of 410 mg. using tablet punches having a diameter of approximately three-eighths inch. (Tablets may be either flat or biconvex and may be scored if desired.)

Example 8 Tablet Formulation Per Tablet Antibiotic l745A/X mg Dicalcium Phosphate Dihydratc, Unmilled 175 mg Corn Starch 24 mg Magnesium Stearate 1 mg Total Weight 225 mg l. Antibiotic l745A/X was mixed with lactose and corn starch in a suitable mixer.

2. The mixture was further blended by passing through a Fitzpatrick Comminuting Machine with a N0. 1A screen with knives forward.

3. The blended powder was returned to the mixer, the tale added and blended thoroughly.

4. The mixture was filled into No. 4 hard shell gelatin capsules on a Parke Davis capsulating machine.

Example 10 Tablet Formulation Per Tablet Antibiotic l745A/X 200 mg Dicalcium Phosphate Dihydratc. Unmillcd 235 mg Corn Starch mg FD&C Yellow No.5 Aluminum Lake 257: 2 mg Durkec 117 25 mg Calcium Stearatc 3 mg Total Weight 535 mg PROCEDURE:

1. All the ingredients were mixed thoroughly and Fitzed (Model D) using a N0. 1A screen, medium speed.

2. The mixture was remixed and slugged.

3. The slugs were screened on an Oscillator through a No. 14 mesh screen and compressed on an E" machine.

1. All of the ingredients were mixed until thoroughly blended in a suitable size container.

2. The powder was filled into No. 2, two-piece, hard shell gelatin capsules to an approximate fill weight of 350 mg. using a Parke Davis capsulating machine. (Any similar type machine may be used).

We claim:

1. The compound of the formula 2. The compound of claim 1 which is erythronolide A 

1. THE COMPOUND OF THE FORMULA
 2. The compound of claim 1 which is erythronolide A oxime. 